During the past decade a number of drugs which are of therapeutic importance in treating various forms of cardiovascular disease have been developed and have been shown to exert their clinical effects by blocking voltage dependent calcium channels. There are several chemical classes of drugs which seem to act in somewhat different pharmacologic mechanisms. However, they all have in common the ability to block calcium channels. In the United States examples of three chemical classes of the drugs which are marketed commercially are respectively nifedipine, verapamil and diltiazem. These drugs are used extensively for treating angina pectoris and hypertension, but have also been evaluated for potential utility in treating numerous other medical conditions.
Dosage requirements for these drugs can vary considerably among different patients. In part the variable dose requirement is related to differences in absorption and metabolism of the drug among individuals. Attaining the optimal doses is important in securing maximal therapeutic benefit and avoiding potentially serious side effects of these drugs. It is generally felt that a simple and sensitive technique to measure these drugs in blood and other body tissues would facilitate selecting optimal doses.
Only three organic calcium antagonist drugs are currently marketed in the United States. Several others are already marketed in Europe and it is likely that within a few years there will be a substantial number in use throughout the world. Ideally, methods for measuring levels of these drugs in body fluids should be applicable to all the drugs. It is also possible that metabolites of some of the drugs may account for pharmacologic activity of the parent drug and so an ideal method should be able to measure pharmacologically active but not inactive metabolites in addition to the parent drug.
Presently available techniques include high performance liquid chromatography and radioimmunoassay. None of these techniques is used routinely in the clinic because of various technical diffuculties. Most of these techniques are applicable for individual drugs rather than for the whole class of calcium antagonists. Also such prior methods do not detect active metabolites.
In the past few years it has become possible to measure receptor sites where the organic calcium antagonist drugs exert their therapeutic actions. Receptors are measured by monitoring the binding of the radiolabeled form of a calcium antagonist drug to membranes from tissues that possess these receptors, including brain, heart, and skeletal muscle. Radioactive drugs that have been used to lable calcium antagonist receptors include .sup.3 H-nitrendipine (Murphy and Snyder, Eur. J. Phamacol., 77:201-202, 1982; Gould, Murphy and Snyder, Proc. Natl. Acad. Sci. USA, 79:3656-3660, 1982; Bolger et al, Biochem. Biophys. Res. Comm., 104:1604-1609, 1982; Ehlert et al., Life Sci., 30:219-220, 1982; Belleman et al., Drug Res., 32:361-363 1982) and .sup.3 H-nimodipine (Belleman et al., Drug Res., 32:361-363, 1982). While the dihydropyridine calcium antagonists such as nifedipine compete directly for .sup.3 H-nitrendipine binding, drugs of other calcium antagonists classes, such as verapamil and diltiazem, interact allosterically with the same receptor site and also reduce .sup.3 H-nitrendipine binding (Yamamura et al., Biochem. Biophys. Res. Comm., 108:640-646, 1982; Murphy, Gould, Largent and Snyder, Proc. Natl. Acad. Sci., USA, 1983, in press).
None of these publications or any other publications describing receptor binding for calcium antagonist drugs has disclosed anything beyond the fact that receptor sites for the calcium antagonist drugs can be measured with various radioactive forms of these drugs and that the drugs compete with the binding of these radioactive agents for the receptor.
Moreover, the information contained in the above mentioned publications does not provide a tool for measuring amounts of the calcium antagonist drugs in body fluids of human patients, because of a number of needed elements, all of which were yet to be discovered, had to be discovered for a successful assay for levels of the calcium antagonist drugs. For a successful assay for a calcium antagonist drug levels it was necessary to discover the nonspecific effects of body fluids on the binding properties of the calcium antagonist receptors and discover means of reducing or abolishing them. It was also necessary to discover that calcium antagonist drugs which added to body fluid could be recovered in a form that could still interact with the receptor sites. It was also necessary to show that in the presence of body fluids increasing amounts of the calcium antagonist drugs would in a predictable fashion produce progressively greater blockade of these receptors. Only after making a series of discoveries as disclosed herein, which reduced nonspecific effects of body fluids on the calcium antagonist receptors, permitted recovery of added calcium antagonist drugs and resulted in reproducible augmentations in receptor blockade with increasing amounts of calcium antagonist drugs in body fluids, was it possible to measure calcium antagonist drugs in body fluids with this invention.